scholarly journals A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP

2008 ◽  
Vol 190 (15) ◽  
pp. 5178-5189 ◽  
Author(s):  
Linda M. Holland ◽  
Sinéad T. O'Donnell ◽  
Dmitri A. Ryjenkov ◽  
Larissa Gomelsky ◽  
Shawn R. Slater ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Cyclase Activity ◽  
Mrna Levels ◽  
Diguanylate Cyclase ◽  
Diguanylate Cyclases ◽  
Exopolysaccharide Biosynthesis ◽  
Ggdef Domain ◽  
Domain Protein ◽  
Biofilm Development

ABSTRACT Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

scholarly journals Indirect Modulation of the Intracellular c-Di-GMP Level inShewanella oneidensisMR-1 by MxdA

2011 ◽  
Vol 77 (6) ◽  
pp. 2196-2198 ◽  
Author(s):  
Shauna Rakshe ◽  
Maija Leff ◽  
Alfred M. Spormann
Keyword(s):  
Biofilm Formation ◽  
Shewanella Oneidensis ◽  
Cellular Level ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Ggdef Domain ◽  
Domain Protein

ABSTRACTThe GGDEF domain protein MxdA, which is important for biofilm formation inShewanella oneidensisMR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP inS. oneidensis, it modulates the c-di-GMP pool indirectly.

scholarly journals BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

2007 ◽  
Vol 189 (22) ◽  
pp. 8165-8178 ◽  
Author(s):  
Sherry L. Kuchma ◽  
Kimberly M. Brothers ◽  
Judith H. Merritt ◽  
Nicole T. Liberati ◽  
Frederick M. Ausubel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Intracellular Signaling ◽  
Biofilm Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Phosphodiesterase Activity ◽  
Swarming Motility ◽  
Ggdef Domain

ABSTRACT The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

scholarly journals The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

2021 ◽  
Author(s):  
Roshni R. Kharadi ◽  
Kayla Selbmann ◽  
George W. Sundin
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Erwinia Amylovora ◽  
Initial Surface ◽  
Diguanylate Cyclase ◽  
Wide Range ◽  
Biofilm Development ◽  
Bacterial Systems

AbstractCyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system.SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

scholarly journals Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain

2010 ◽  
Vol 76 (24) ◽  
pp. 8160-8173 ◽  
Author(s):  
Shuwen An ◽  
Ji'en Wu ◽  
Lian-Hui Zhang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Phosphodiesterase Activity ◽  
Low Oxygen ◽  
Ggdef Domain ◽  
Biofilm Dispersal ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

scholarly journals Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae

2021 ◽  
Vol 9 (3) ◽  
pp. 495
Author(s):  
Weiwei Yan ◽  
Yiming Wei ◽  
Susu Fan ◽  
Chao Yu ◽  
Fang Tian ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Binding Activity ◽  
Xanthomonas Oryzae ◽  
Bacterial Cells ◽  
Diguanylate Cyclase ◽  
Biological Functions ◽  
Swimming Motility ◽  
Ggdef Domain ◽  
Domain Protein ◽  
Sliding Motility

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

scholarly journals Identification and Characterization of a Phosphodiesterase That Inversely Regulates Motility and Biofilm Formation in Vibrio cholerae

2010 ◽  
Vol 192 (18) ◽  
pp. 4541-4552 ◽  
Author(s):  
Xianxian Liu ◽  
Sinem Beyhan ◽  
Bentley Lim ◽  
Roger G. Linington ◽  
Fitnat H. Yildiz
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Lifestyle Changes ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Ggdef Domain

ABSTRACT Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 (cdgJ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

scholarly journals Effects of CO2laser irradiation on matrix-rich biofilm development formation–an in vitro study

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2458 ◽  
Author(s):  
Bruna Raquel Zancopé ◽  
Vanessa B. Dainezi ◽  
Marinês Nobre-dos-Santos ◽  
Sillas Duarte ◽  
Vanessa Pardi ◽  
...  
Keyword(s):  
Contact Angle ◽  
Biofilm Formation ◽  
Dental Enamel ◽  
In Vitro Study ◽  
Dry Weight ◽  
Vitro Study ◽  
Enamel Surface ◽  
Control Group ◽  
Biofilm Development

BackgroundA carbon dioxide (CO2) laser has been used to morphologically and chemically modify the dental enamel surface as well as to make it more resistant to demineralization. Despite a variety of experiments demonstrating the inhibitory effect of a CO2laser in reduce enamel demineralization, little is known about the effect of surface irradiated on bacterial growth. Thus, this in vitro study was preformed to evaluate the biofilm formation on enamel previously irradiated with a CO2laser (λ = 10.6 µM).MethodsFor this in vitro study, 96 specimens of bovine enamel were employed, which were divided into two groups (n = 48): 1) Control-non-irradiated surface and 2) Irradiated enamel surface. Biofilms were grown on the enamel specimens by one, three and five days under intermittent cariogenic condition in the irradiated and non-irradiated surface. In each assessment time, the biofilm were evaluated by dry weigh, counting the number of viable colonies and, in fifth day, were evaluated by polysaccharides analysis, quantitative real time Polymerase Chain Reaction (PCR) as well as by contact angle. In addition, the morphology of biofilms was characterized by fluorescence microscopy and field emission scanning electron microscopy (FESEM). Initially, the assumptions of equal variances and normal distribution of errors were conferred and the results are analyzed statistically by t-test and Mann Whitney test.ResultsThe mean of log CFU/mL obtained for the one-day biofilm evaluation showed that there is statistical difference between the experimental groups. When biofilms were exposed to the CO2laser, CFU/mL and CFU/dry weight in three day was reduced significantly compared with control group. The difference in the genes expression (Glucosyltransferases (gtfB) and Glucan-binding protein (gbpB)) and polysaccharides was not statically significant. Contact angle was increased relative to control when the surface was irradiated with the CO2laser. Similar morphology was also visible with both treatments; however, the irradiated group revealed evidence of melting and fusion in the specimens.ConclusionIn conclusion, CO2laser irradiation modifies the energy surface and disrupts the initial biofilm formation.

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